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WO1990011370A1 - Sonde de polynucleotide, procede et kit d'identification et de detection de bacteries gram-negatives - Google Patents

Sonde de polynucleotide, procede et kit d'identification et de detection de bacteries gram-negatives Download PDF

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Publication number
WO1990011370A1
WO1990011370A1 PCT/NL1990/000033 NL9000033W WO9011370A1 WO 1990011370 A1 WO1990011370 A1 WO 1990011370A1 NL 9000033 W NL9000033 W NL 9000033W WO 9011370 A1 WO9011370 A1 WO 9011370A1
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WIPO (PCT)
Prior art keywords
probe
gram
species
bacteria
probes
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PCT/NL1990/000033
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English (en)
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Johannes Petrus Maria Tommassen
Aldegonda Maria Johanna Spierings
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Holland Biotechnology Bv
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Publication date
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Publication of WO1990011370A1 publication Critical patent/WO1990011370A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a polynucleotide probe which speci ⁇ fically recognizes a genus or species of Gram-negative bacteria.
  • a rapid determination of microorganisms is of great impor- tance in medicine and health care also, for example for localizing and combating infections. Furthermore, there is also a need for rapid and effective methods of determination for the presence of bacteria in the environmental sector.
  • the microorganism is subjected to a large series of tests in each of which it is determined whether or not a specific enzyme activity occurs. The combination of activities found gives an indication for the species of bacteria or for the genus of bacteria in question. Methods of this type are laborious and, above all, not definitive: a bacterium or group of bacteria is detected only with a specific probability (of less than 100%).
  • DNA-DNA hybri ⁇ dization is regarded as a highly promising technique (see Klausner and Wilson, Bio-Technology 1_, 471-478, 1983).
  • a DNA fragment which is specific for a species of bac ⁇ teria or a group of bacteria is sought as a "probe" for structural ⁇ ly related DNA in bacteria which it is desired to identify.
  • the great advantage of a detection technique of this type is that testing is for the genotype instead of, as customary, for the phenotype.
  • the route which has been followed up to now for developing DNA probes for bacteria is the fragmentation of bacterial DNA in a relatively arbitrary manner, cloning fragments in plasmid vectors and investigating, with the aid of radioactive markers, whether fragments hybridize specifically with bacterial DNA.
  • the sequence of such frag- ent ⁇ must then be determined, after which oligonucleotides are prepared and tested again.
  • the disadvantage of probes of this type is that they demand a prolonged and laborious preparation, a prepa ⁇ ration which, moreover, must be developed entirely anew for a subsequent species of bacteria to be investigated, as a result of which the composition of a kit for determining various bacteria in samples on the basis of such probes is virtually impossible.
  • Probes based on ribosomal RNA are also known: however, in a number of cases these display a troublesome cross-reactivity (inadequate inclusivity and exclusivity). It has been found that certain sequences of genes which code for outer membrane proteins of Gram-negative bacteria and the sequences complementary hereto are very suitable as a poly ⁇ nucleotide probe with which microorganisms of a specific species or genus can be detected, for example in foodstuffs, in a clinical specimen or in the environment.
  • the polynucleotide probe according to the invention therefore contains a polynucleotide which corresponds with or is complementa ⁇ ry to a fragment of a gene or messenger RNA coding for an outer membrane protein of a Gram-negative bacterium.
  • An advantage of the polynucleotide probe according to the invention is that this contains only a relatively small number of nucleotides (of the order of 10-40 base pairs), as a result of which this can be simply prepared and used. At the same time, this probe proves to be specific for a certain genus or species of Gram- negative bacteria.
  • the polynucleotide probe can contain either a polydeoxyribo- nucleotide (DNA probe) or a polyribonucleotide (RNA probe).
  • DNA probe polydeoxyribo- nucleotide
  • RNA probe polyribonucleotide
  • polynucleotide does not indicate a specific minimum number of nucleic acids per molecule and this term is therefore not in absolute contrast to "oligonucleotide”.
  • a further advantage of the polynucleotide probe according to the invention is that the hybridization can also be made detectable by other methods, for example by the so-called PCR (polymerase chain reaction) method. Furthermore, the probes are relatively easy to combine into a kit with which a series of relevant Gram-negative bacteria can be identified, for example in foodstuffs, body fluid, water samples and the like.
  • Outer membrane proteins are proteins which are present in the outer membrane of the cell envelope of Gram-negative bacteria, where they fulfil various functions such as allowing the passage of nutrients (the pore-forming outer membrane proteins or porines) and of larger dissolved substances and contributing to the structure and the binding of the outer membrane. These proteins are desig ⁇ nated OmpA, OmpC, OmpF, PhoE, LamB, Tsx, OmpT, FhuA, BtuB, FepA, FecA, etc.
  • the heterologous fragments of the outer membrane proteins frequently occur in the parts of the outer membrane proteins which are exposed at the cell surface, while the membrane-bridging parts are substantially preserved. Therefore, the fragments of the genes which correspond to the exposed fragments of the outer membrane proteins are particularly suitable as a basis for specific poly ⁇ nucleotide probes according to the invention.
  • An example of a suitable probe for detecting Salmonella species is a probe which contains the 23-mer oligodeoxynucleotide TTTAGTAGACGGGCCGCCAGGGA, corresponding to an exposed part of the OmpA protein of S. typhimurium.
  • the probes according to the invention can be built up on the basis of the outer membrane proteins, in principle of all Gram- negative bacteria, and therefore are used for detecting the same Gram-negative species or genera of bacteria.
  • Probes for Enterobac ⁇ teriaceae are particularly important because these bacteria play a major role in infections, food poisoning, biological contamination and the like. Bacteria of the genera Enterobacter, Klebsiella, Salmonella, Yersinia, Edwardsiella, Erwinia, Serratia, Citrobacter, Proteus and Providencia may be mentioned.
  • the invention can also be used for detecting Gram-negative bacteria other than Enterobac ⁇ teriaceae, such as Pseudomonas.
  • the polynucleotide probes according to the invention recog ⁇ nize specific genera of bacteria and possibly species of bacteria.
  • a probe derived from an outer membrane protein of Salmonella typhimurium proves to recognize all Salmonella species and not to show any false-positive reaction with other species.
  • the current categorization of bacteria in genera and species is by no means perfect, as a result of which it can arise that a probe derived from a species or strain belonging to a specific genus does not recognize a species with the same genus name.
  • a probe derived, for example, from an Escherichia coli also recognizes Shigella sp., which confirms that E. coli and Shigella sp. belong to the same genus and perhaps even to a single species (see W.J. Brenner in Bergey's Manual of Systematic Bacte ⁇ riology, vol. I, pp. 410-411, Williams & Williams Co. Baltimore (1984)).
  • the fact that the polynucleotide probes according to the invention have such a high exclusivity and inclusivity is particu ⁇ larly surprising in view of the results with DNA probes according to the prior art.
  • the polynucleotide probe according to the invention can be prepared on the basis of a suitable fragment of a gene or messenger RNA coding for an outer membrane protein of the chosen bacterium by lining up together the relevant nucleotides according to a proce ⁇ dure known per se.
  • the preparation can advantageously be automated using a DNA synthesis installation.
  • the ability of the probe thus obtained selectively to detect bacteria is demonstrated, for example, by means of hybridization in a so-called slot-blot procedure (P.J. Carter et al., Cell 38, 835- 840 (1984)).
  • the DNA of the bacterium or bacteria to be investigated is liberated from the cells and attached to a filter and then brought into contact with a probe according to the invention, provided with, for example, a radioactive label. After hybridization it is investigated, for example by means of an auto- radiogram, whether the sample has reacted with a nucleotide sequence, in other words has recognized this nucleotide sequence.
  • PCR polymerase chain reaction
  • Another recently developed procedure which can be used to detect hybridization is the coupling to viral RNA, after which the probe is hybridized with the DNA sample to be determined. Any hybridization can then be demonstrated by in vitro reproduction of the hybridized RNA with the aid of viral replicas. In this way such a large amount of the hybridized RNA can be obtained in a short time that detection without radioactive labelling is pos- sible. See also P.M. Lizardi et al., Biotechnology ⁇ _, 1197-1102 (1988).
  • the polynucleotide probe can be used selectively to detect a species or genus of bacteria within a multiplicity of bacteria, for example in foodstuffs.
  • the polynucleotide probe according to the invention is also very suitable for identifying isolated bacteria, where appropriate in a pure culture, for medical use (infections).
  • the invention therefore also relates to a method for the detection and identification of Gram-negative bacteria, in parti ⁇ cular of Enterobacteriaceae, using the polynucleotide probe- as described above.
  • the method consists, for example, in isolating the poly ⁇ nucleotide material from the sample to be investigated, or attaching it to a carrier, in a known manner and then bringing it into contact with a probe according to the invention.
  • the hybri- dization can then be detected by various methods, for example by using 32 P-ATP, viral replicas etc.
  • the method according to the invention is particularly advantageous when using the PCR method as indicated above, in which case the polynucleotide fragment repro ⁇ quizd can be detected in various ways.
  • the invention also relates to a kit for the selective detection/identification of Gram-negative bacteria, which kit contains one or more of the polynucleotide probes described above.
  • this kit contains a combination of DNA probes for testing for the presence of a series of Enterobacteriaceae.
  • the kit according to the invention can also contain means for isolating the (hybridized) polynucleotide, such as filters, for detecting the hybridization signal, such as labelling substances, and for in vitro reproduction of hybridized polynucleotide fragments, such as a DNA-polymerase or a viral polymerase, and also other means for carrying out an iden ⁇ tification of bacteria, and if desired a method for this.
  • the examples below illustrate that polynucleotide sequences which code for parts of outer membrane proteins exposed at the cell surface, or the sequences complementary to these, can be used as genus-specific probes for the identification of Gram-negative bacteria.
  • the oligodeoxynucleotides are prepared automatically with the aid of a Biosearch 8600 DNA synthesizer and are then purified by means of high pressure liquid chromatography.
  • the oligomers are labelled at the 5'-end by enzymatically catalyzed transfer of 32 P phosphate from [ ⁇ - 3*** P]ATP (3000 Ci/mmol, Amersham Int. pic, Amersham, England) with T4 polynucleotide kinase (Pharmacia, Uppsala, Sweden) using the method of Maniatis et al., Molecular Cloning, a laboratory manual, Cold Spring Harbor, N.Y. (1982). Bacterial strains and plasmids
  • E. coli B and C Institute for Molecular Biology and Medical Biotechnology, University of Utrecht).
  • coli strain S1, S2, S3, S4, S5, S6, S8 and S9 isolated from blood cultures of patients having bacteremia, F1, F5, F6, F9 and F12 isolated from faeces of healthy volunteers, and U6, U7, U8, U11 and U13 isolated from virine of patients having urinary tract infections, are described by Overbeeke and Lugtenberg (J. Gen. Microbiol. ⁇ 21373-380 (1980).
  • Strain EIEC an entero- invasive E.
  • enterobacterial strains used were Edwardsiella tarda, Enterobacter aerogenes, Citrobacter freundii, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, Salmonella braenderup, Salmonella derby, Salmonella panama, Serratia marcescens, Shigella flexneri, Shigella sonnei (H. Hofstra, J. Dankert, J. Gen. Microbiol. 119, 123-131 (1980), Salmonella typhimurium 8J2353 (T. Sato, T. Yura, J. Bacteriol.
  • Salmonella typhimurium LT a Salmonella typhimurium B7121-2 pro
  • Salmonella typhimurium KB1711 K. Bauer et al., J. Bacterol. .161.: 813-816 (1985)
  • the following were used for the polymerase chain reaction procedure: E. coli K-12 strain CGSC4234 (E. coli Genetic Stock Center, Department of Human Genetics, Yale University School of Medicine, New Haven, Conn.), Salmonella panama and Salmonella typhimurium SJ2353.
  • pACYC184 A. Chang, S. Cohen, J. Bacteriol. J_34: 1141-1156. (1978)
  • pJP29 contains the phoE gene of E. coli, D. Bosch et al., J. Mol. Biol. 189, 449-455 (1986)
  • pKP 2 contains the phoE gene of Klebsiella pneumoniae, P. van der Ley et al., Eur. J. Biochem. 164: 469-475 (1987)
  • pEC17 contains the phoE gene of Enterobacter cloacae, C.
  • Verhoef et al. Gene 32: 107-115 (1984), plasmid pBR322 (F. fiolivar et al., Gene 2 : 95-113 (1977)) and its derivative pSTl (contains a portion of the phoE gene of Salmonella typhimurium, G. Spierings, not published).
  • NC nitrocellulose
  • the filters were pre-hybridized for 45 minutes at 60°C in 0.25% Protifar (Nutricia NV, Zoetermeer), 6 x SSC (90 mM sodium chloride, 90 mM sodium citrate pH 7.0). After adding the probe (10 pmol DNA), the filter was hybridized for 1.5 hours at 60 ⁇ C. The filters were washed twice with 6 x SSC at 60°C and autoradiograms of the filters were made by exposure to a Fuji X-ray film for 3 days. DNA spot procedure: 1 ⁇ l of the plasmid solutions ( « 0.5 ⁇ g/ ⁇ l) was spotted on NC. The filters were then treated and hybridized as described in the slot-blot hybridization procedure.
  • ST1/ST3c and ST2/ST3c were used as primer couples, resulting in the reproduction of fragments of, respectively, 285 and 156 base pairs.
  • 40 cycles of 1 minute at 92 ⁇ C, 2 minutes at 42 ⁇ C and 2 minutes at ⁇ '-'C were run with the aid of a programmable incubator block (PHC-1, New Brunswick Scientific B.V., Soest, Holland). 10 ⁇ l of the reaction mixture was analysed on a 1.3% agarose gel.
  • Salmonella probes Three Salmonella probes were synthesized, based on the first, the second and the third fragment of the OmpA protein of S. typhimurium exposed at the cell surface. The DNA sequences of these fragments are indicated as ST1 , ST2 and ST3c respectively in Figure 1. The inclusivity and exclusivity of ST3c was tested in a slot-blot hybridization procedure. Figure 4 shows the autoradiogram of the filter after hybridization with the probe. The following strains were applied to the filter: 1A, P. mirabilis; 1B, S. panama; 1C, S. typhimurium; 2A, S. marcescens; 2B, C. freundii, 2C, K. pneumoniae; 3A, E.
  • the probe does not react with the other Enterobacte ⁇ riaceae. However, under the test conditions used the probe does not react with S. panama. Under the conditions used in the PCR reaction ( Figure 5), however, both the chromosomal DNA isolated from S. panama (Slots 5 and 6) and the chromosomal DNA isolated from S. typhimurium (Slots 3 and 4) were found to be used as substrate. When the chromosomal DNA isolated from E. coli was offered as substrate, amplification of the fragment of 285 base pairs (Slot 1) also took place when the probes ST. and ST3c were used, and also of the fragment of 156 base pairs (Slot 2) when the probes ST2 and ST3c were used.
  • Example II Shigella probe For the development of the Shigella probe, use was made of the very close relationship which exists between Shigella and E. coli (see W.J. Brenner, Family I Enterobacteriaceae in: Bergey's Manual of Systematic Bacteriology, vol. I, The Williams & Wilkins Co., Baltimore, pp. 410-411 (1984)). It was therefore assumed that the sequences of the phoE genes of Shigella species are virtually identical to the known sequence of E. coli K-12 phoE gene. The Shigella probe for which the sequence is shown in Figure 2 together with the corresponding sequences of K. pneumoniae and E. cloacae (P.
  • van der Ley et al. is based on the fifth portion of the PhoE protein of E. coli exposed at the cell surface.
  • the specificity of the probe was tested with the aid of a slot-blot hybridization procedure.
  • the autoradiogram is shown in Fig. 6.
  • the following strains were applied to the filter: 1A, P. mirabilis; 1B, S. panama; 1C, S ⁇ typhimurium; 2A, S. marcescens; 2B, C. freundii; 2C, K. pneumoniae; 3A, E. tarda; 3B, E. aerogenes; 3C, P.
  • the probe was tested on a number of E. coli strains isolated from blood cultures of patients having bacte- remia, from faeces of healthy volunteers and from urine of patients having urinary tract infections. These strains represent a variety of 0 and K serotypes (as described by Overbeeke and Lugtenberg, J.
  • Figure 7 shows that the probe reacted with all these different strains.
  • the Enterobacter probe and the Klebsiella probe are based on the eighth fragment of the PhoE protein of, respectively, E. cloacae and K. pneumoniae exposed at the cell surface.
  • the specificity of the probes was investigated in DNA spot tests.
  • the autoradiogram of the filter hybridized with the Enterobacter probe and hybridized with the Klebsiella probe is shown in Figure 8 on the left-hand photograph and right-hand photograph respectively.
  • the following plasmids were applied to the filters: 1A, pEC17; 1B, pBR322; pJP29; 2B, pACYC184;
  • Klebsiella probe reacts only with pKP 2 .

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Abstract

L'invention concerne une sonde de polynucléotides (ADN ou ARN) reconnaissant un gène ou une espèce de bactérie gram-négatives, notamment d'un gène ou d'une espèce d'Enterobacteriaceae. Ladite sonde correspond à un fragment relativement court d'une protéine de membrane extérieure de la bactérie et est hautement spécifique (exclusive et inclusive) au gène ou à l'espèce étudiés. La sonde destinée à différents genres ou à différentes espèces est utilisée dans un procédé de contrôle de contamination bactérienne, par exemple dans des produits alimentaires ou dans des fluides biologiques, et peut se présenter sous la forme d'un kit de diagnostic.
PCT/NL1990/000033 1989-03-17 1990-03-16 Sonde de polynucleotide, procede et kit d'identification et de detection de bacteries gram-negatives WO1990011370A1 (fr)

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NL8900670A NL8900670A (nl) 1989-03-17 1989-03-17 Polynucleotide-probe, werkwijze en kit voor het identificeren en opsporen van gram-negatieve bacterien.
NL8900670 1989-03-17

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JP (1) JPH05504672A (fr)
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WO (1) WO1990011370A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0479117A1 (fr) * 1990-10-05 1992-04-08 F. Hoffmann-La Roche Ag Méthodes et réactifs pour l'identification bactérienne
EP0669989A4 (fr) * 1991-08-22 1994-12-20 Univ Washington Sondes de polynucleotide de detection de salmonelle.
LT3828B (en) 1993-11-10 1996-03-25 Fermentas Biotech Inst Process for detecting salmonella bacteries and probe for the same
US5620847A (en) * 1990-10-05 1997-04-15 Hoffman-La Roche Inc. Methods and reagents for detection of bacteria in cerebrospinal fluid
US5635348A (en) * 1990-10-05 1997-06-03 Hoffmann-La Roche Inc. Method and probes for identifying bacteria found in blood
US5656740A (en) * 1994-06-06 1997-08-12 E. I. Du Pont De Nemours And Company Nucleic acid fragments useful in the detection of Salmonella
US5747257A (en) * 1996-02-29 1998-05-05 E. I. Du Pont De Nemours And Company Genetic markers and methods for the detection of escherichia coli serotype-0157:H7
WO1998018958A1 (fr) * 1996-10-28 1998-05-07 Compagnie Gervais Danone Procede de mise en evidence de contaminants microbiologiques vivants dans un echantillon de produit a usage alimentaire
US5922538A (en) * 1996-11-08 1999-07-13 E.I. Du Pont De Nemours And Company Genetic markers and methods for the detection of Listeria monocytogenes and Listeria spp
EP0652291A4 (fr) * 1992-07-07 1999-08-18 Fuso Pharmaceutical Ind Sonde pour diagnostiquer des maladies infectieuses.
US6790661B1 (en) 1999-07-16 2004-09-14 Verax Biomedical, Inc. System for detecting bacteria in blood, blood products, and fluids of tissues
WO2005090596A3 (fr) * 2004-03-19 2005-11-24 Univ Catholique Louvain Dosage pour la detection et l'identification de micro-organismes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0297291A2 (fr) * 1987-06-03 1989-01-04 BEHRINGWERKE Aktiengesellschaft Protéine de la membrane extérieure F de Pseudomonas aeruginosa

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0297291A2 (fr) * 1987-06-03 1989-01-04 BEHRINGWERKE Aktiengesellschaft Protéine de la membrane extérieure F de Pseudomonas aeruginosa

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Applied and Environmental Microbiology, Volume 55, No. 12, December 1989, American Society for Microbiology, (Washington, D.C., US), G. SPIERINGS et al.: "Development of Enterobacterium-Specific Oligonucleotide Probes based on the Surface-Exposed Regions of outer Membrane Proteins", pages 3250-3252 *
Journal of Bacteriology, Volume 149, January 1982, (Washington, D.C., US), S.T. COLE et al.: "Cloning and Expression in Escherichia Coli K-12 of the Genes for Major outer Membrane Protein ompA from Shigella Dysenteriae, Enterobacter Aerogenes, and Serratia Marcescens", pages 145-150 *
Journal of Clinical Microbiology, Volume 18, No. 1, July 1983, American Society for Microbiology, (Washington, D.C., US), A.M. PALVA: "OmpA Gene in Detection of Escherichia Coli and other Enterobacteriaceae by Nucleic Acid Sandwich Hybridization", pages 92-100 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5635348A (en) * 1990-10-05 1997-06-03 Hoffmann-La Roche Inc. Method and probes for identifying bacteria found in blood
EP0479117A1 (fr) * 1990-10-05 1992-04-08 F. Hoffmann-La Roche Ag Méthodes et réactifs pour l'identification bactérienne
US5620847A (en) * 1990-10-05 1997-04-15 Hoffman-La Roche Inc. Methods and reagents for detection of bacteria in cerebrospinal fluid
EP0669989A4 (fr) * 1991-08-22 1994-12-20 Univ Washington Sondes de polynucleotide de detection de salmonelle.
EP1160334A3 (fr) * 1992-07-07 2004-01-02 Fuso Pharmaceutical Industries Ltd. Sonde pour diagnostiquer des maladies infectieuses liées à pseudomonas aeruginosa
EP0652291A4 (fr) * 1992-07-07 1999-08-18 Fuso Pharmaceutical Ind Sonde pour diagnostiquer des maladies infectieuses.
EP1167544A3 (fr) * 1992-07-07 2004-03-03 Fuso Pharmaceutical Industries Ltd. Sonde pour diagnostiquer des maladies infectieuses à cause d' Escherichia coli, Klebsiella pneumoniae ou Enterobacter cloacae
LT3828B (en) 1993-11-10 1996-03-25 Fermentas Biotech Inst Process for detecting salmonella bacteries and probe for the same
US5656740A (en) * 1994-06-06 1997-08-12 E. I. Du Pont De Nemours And Company Nucleic acid fragments useful in the detection of Salmonella
US5660981A (en) * 1994-06-06 1997-08-26 E. I. Du Pont De Nemours And Company Selection of diagnostic genetic markers in microorganisms and use of a specific marker for detection of salmonella
US5747257A (en) * 1996-02-29 1998-05-05 E. I. Du Pont De Nemours And Company Genetic markers and methods for the detection of escherichia coli serotype-0157:H7
WO1998018958A1 (fr) * 1996-10-28 1998-05-07 Compagnie Gervais Danone Procede de mise en evidence de contaminants microbiologiques vivants dans un echantillon de produit a usage alimentaire
US6472149B1 (en) 1996-10-28 2002-10-29 Compagnie Gervais Danone Method for detecting live microbiological contaminants in a food product sample
US5922538A (en) * 1996-11-08 1999-07-13 E.I. Du Pont De Nemours And Company Genetic markers and methods for the detection of Listeria monocytogenes and Listeria spp
US6790661B1 (en) 1999-07-16 2004-09-14 Verax Biomedical, Inc. System for detecting bacteria in blood, blood products, and fluids of tissues
WO2005090596A3 (fr) * 2004-03-19 2005-11-24 Univ Catholique Louvain Dosage pour la detection et l'identification de micro-organismes
US7785780B2 (en) 2004-03-19 2010-08-31 Universite Catholique De Louvain Assay for detecting and identifying micro-organisms

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NL8900670A (nl) 1990-10-16
EP0464123A1 (fr) 1992-01-08
JPH05504672A (ja) 1993-07-22
AU5351790A (en) 1990-10-22

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